Sequence-specific DNA binding proteins, more commonly known as transcription factors, represent a group of molecules within the cell that function to connect extracellular signals to intracellular responses by directly affecting gene transcription. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind consensus regions in the promoters of various genes and activate the transcription of the respective target gene.
The Y-box binding protein family of transcription factors, so named because of the consensus sequence to which they bind (the Y-box), are the most conserved nucleic acid binding proteins found in bacteria, plants and animals (Wolffe, BioEssays, 1994, 16, 245-251). The Y-box, comprising an inverted CCAAT sequence, has been identified in over 170 promoters to date and Y-box proteins mediate the transcription of many of these genes (Mantovani, Nucleic Acids Res., 1998, 26, 1135-1143).
All Y-box proteins are structurally and functionally organized into three distinct domains, an N-terminal domain rich in proline and alanine which acts as a transcriptional regulator, a central cold shock domain (CSD) which binds to the Y-box in the promoter of many genes and a C-terminal domain containing alternatively charged positive and negative amino acids that is thought to mediate protein-protein interactions (Graumann and Marahiel, Trends Biochem. Sci., 1998, 23, 286-290; Wolffe, BioEssays, 1994, 16, 245-251).
Although the modular CSD domain is highly conserved across species, these proteins have a broad specificity for nucleic acids, binding double-stranded DNA, damaged DNA, single-stranded DNA and RNA and have been shown to operate on both the transcriptional and translational levels (Hasegawa et al., Nucleic Acids Res., 1991, 19, 4915-4920; Ladomery and Sommerville, Nucleic Acids Res., 1994, 22, 5582-5589).
Eukaryotic Y-box binding protein 1 (also known as YB-1 and DNA binding protein B or dbpB) is the best characterized Y-box protein and was originally identified using a radiolabeled double-stranded DNA Y-box sequence to screen a .lambda.gt11 expression cDNA library for transcription factors that associate with the Y-box (Didier et al., Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 7322-7326; Sakura et al., Gene, 1988, 73, 499-507). Recently, two new alleles of the Y-box binding protein 1, one of which was isolated from a patient with rheumatoid arthritis, were identified (Singal and Miller, Gene, 1995, 154, 299-300). The present invention provides compositions and methods for modulating Y-box binding protein 1 expression, including modulation of the alternate alleles of Y-box binding protein 1.
In studies designed to examine the expression of the major histocompatibility complex (MHC) class II genes, Didier et al. demonstrated that Y-box binding protein 1 binds to the promoter and is a negative regulatory of MHC gene expression.
Other studies of MHC expression confirm the role of Y-box binding protein 1 as a repressor of interferon-.gamma. activation of MHC genes and suggest a model by which Y-box binding protein 1 binds single-stranded regions of DNA within the promoter thereby blocking the binding of other transactivating factors (MacDonald et al., J. Biol. Chem., 1995, 270, 3527-3533; Ting et al., J. Exp. Med., 1994, 179, 1605-1611). The ability of Y-box binding protein 1 to affect MHC gene expression has broad implications in immune regulation.
Y-box binding protein also mediates transcriptional events associated with the development of certain cancers. Studies of the multidrug resistance gene (MDR1) are reported in the art that correlate Y-box binding protein expression and localization with multidrug resistance in osteosarcoma (Oda et al., Clin. Cancer Res., 1998, 4, 2273-2277), breast (Bargou et al., Nat. Med., 1997, 3, 447-450) and epidermal cancers (Koike et al., FEBS Lett., 1997, 417, 390-394; Ohga et al., Cancer Res., 1996, 56, 4224-4228; Ohga et al., J. Biol. Chem., 1998, 273, 5997-6000).
In both osteosarcoma and breast cancers, increased levels of Y-box binding protein 1 correlated with the increased expression of P-glycoprotein, the expression product of the MDR1 gene (Bargou et al., Nat. Med., 1997, 3, 447-450; Oda et al., Clin. Cancer Res., 1998, 4, 2273-2277). Unlike multidrug resistant MCF-7 breast cancer cells, where increased expression and nuclear localization of Y-box binding protein 1 was observed, there was no correlation between localization and histological grade in osteosarcomas.
Studies of head and neck cancers revealed that exposure to ultraviolet radiation increased the nuclear localization of Y-box binding protein 1 in these cells (Koike et al., FEBS Lett., 1997, 417, 390-394). In addition, transient transfection of antisense Y-box binding protein 1 expressing constructs into these cells resulted in decreased Y-box binding protein 1 protein levels and DNA binding activity regardless of ultraviolet irradiation (Ohga et al., J. Biol. Chem., 1998, 273, 5997-6000). These transfectants also demonstrated increased sensitivity to cisplatin and mitomycin C, DNA damaging agents (Ohga et al., Cancer Res., 1996, 56, 4224-4228).
Ise et al. demonstrated, using immunochemical coprecipitation methods that Y-box binding protein 1 interacts in vivo with proliferating cell nuclear antigen (PCNA), an essential protein of DNA repair (Ise et al., Cancer Res., 1999, 59, 342-346).
Taken together, these studies define a role for Y-box binding protein 1 in DNA repair and the sensitization of cells from a diverse array of genotoxic stress signals including agents that induce DNA cross-linking and ultraviolet irradiation.
Y-box binding protein 1 also participates in the regulation of extracellular matrix degradation. Using antisense oligonucleotides transiently transfected or stably expressed via retroviral constructs, Dhalla et al. showed that blocking expression of YB-1b, the chicken homologue of human Y-box binding protein 1, resulted in down-regulation of procollagen .alpha.1(I) gene expression. Considering that the promoter binding site of .alpha.1(I) is conserved between chicken and human, the authors suggest that human Y-box binding protein 1 may mediate the expression of collagen genes (Dhalla et al., Biochem. J., 1998, 336, 373-379).
Others have shown that Y-box binding protein 1 is a major, cell type-specific transactivator of the matrix degrading enzyme, matrix metalloproteinase 2 (MMP2), dtranscription in glomerular mesangial cells. These studies also demonstrated the synergistic interaction of Y-box binding protein 1 with another transcription factor, AP2 (Mertens et al., J. Biol. Chem., 1998, 273, 32957-32965; Mertens et al., J. Biol. Chem., 1997, 272, 22905-22912).
Viral gene expression is also modulated by Y-box binding protein 1. Studies of the JC polyomavirus (JCV), the causative agent of progressive multifocal leukoencephalopathy, demonstrate a critical role for Y-box binding protein 1 in viral transcriptional regulation (Chen et al., Proc. Natl. Acad. Sci. U.S.A., 1995, 92, 1087-1091; Chen and Khalili, J. Virol., 1995, 69, 5843-5848; Safak et al., Mol. Cell. Biol., 1999, 19, 2712-2723). Human T-cell lymphotrophic virus type I (HTLV-I) gene expression is also affected by Y-box binding protein 1 (Kashanchi et al., J. Virol., 1994, 68, 561-565).
Furthermore, it has been suggested that Y-box binding protein 1 may be involved in redox signaling. Studies using antisense oligonucleotides targeting the mouse Y-box binding protein 1 abolished the H.sub.2 0.sub.2 -induced ORE-mediated expression of a reporter linked to the MHC class II HLA-DQ promoter transfected into mouse hepatoma cells (Duh et al., J. Biol. Chem., 1995, 270, 30499-30507).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Y-box binding protein 1. To date, strategies aimed at modulating Y-box binding protein 1 function have involved the use of antibodies, molecules that block upstream entities and antisense expression vectors and oligonucleotides.
However, these strategies are untested as therapeutic protocols and consequently, there remains a long felt need for additional agents capable of effectively inhibiting Y-box binding protein 1 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of Y-box binding protein 1 expression.